U.S. patent application number 09/308424 was filed with the patent office on 2002-10-31 for transgenic mouse expressing the human cyclooxygenase-2 gene and neuronal cell cultures derived therefrom.
Invention is credited to AISEN, PAUL S., PASINETTI, GIULIO M..
Application Number | 20020162130 09/308424 |
Document ID | / |
Family ID | 26709565 |
Filed Date | 2002-10-31 |
United States Patent
Application |
20020162130 |
Kind Code |
A1 |
PASINETTI, GIULIO M. ; et
al. |
October 31, 2002 |
TRANSGENIC MOUSE EXPRESSING THE HUMAN CYCLOOXYGENASE-2 GENE AND
NEURONAL CELL CULTURES DERIVED THEREFROM
Abstract
The present invention relates to the use of nimesulide and
structurally related compounds in the prevention and/or treatment
of neurodegenerative conditions. It is based, at least in part, on
the discovery that nimesulide exhibits a neuroprotective effect
against &bgr.-amyloid induced cell death Without being bound to
any particular theory, it appears that nimesulide inhibits a
non-inflammatory mechanism of neurodegeneration
Inventors: |
PASINETTI, GIULIO M.; (NEW
YORK, NY) ; AISEN, PAUL S.; (POTOMAC, MD) |
Correspondence
Address: |
BAKER & BOTTS
30 ROCKEFELLER PLAZA
NEW YORK
NY
10112
|
Family ID: |
26709565 |
Appl. No.: |
09/308424 |
Filed: |
July 16, 1999 |
PCT Filed: |
November 19, 1997 |
PCT NO: |
PCT/US97/21484 |
Current U.S.
Class: |
800/18 ; 435/325;
435/352; 435/354 |
Current CPC
Class: |
A01K 2217/05 20130101;
C12N 9/0083 20130101; C12N 15/8509 20130101; A01K 2227/105
20130101; A61K 31/18 20130101; C12N 2517/02 20130101; A01K 67/0278
20130101; C12N 2830/008 20130101; A01K 2207/15 20130101; A01K
2217/00 20130101; A01K 2267/03 20130101; A61P 25/28 20180101; A01K
2267/0312 20130101; A01K 2267/0318 20130101; A01K 67/0275
20130101 |
Class at
Publication: |
800/18 ; 435/325;
435/352; 435/354 |
International
Class: |
A01K 067/027; C12N
005/06 |
Claims
What is claimed is:
1. Use of nimesulide for the preparation of a pharmaceutical
composition for treating a neurodegenerative condition.
2. Use of nimesulide for the preparation of a pharmaceutical
composition for treating Alzheimer's Disease.
3. Use of nimesulide for the preparation of a pharmaceutical
composition for treating epilepsy.
4. Use of nimesulide for the preparation of a pharmaceutical
composition for treating amyotrophic lateral sclerosis.
5. Use of nimesulide for the preparation of a pharmaceutical
composition for treating a neurodegenerative condition in a
subject, wherein the amount of nimesulide present in the
pharmaceutical composition results in exposure of the affected
neurons to a local concentration of nimesulide of one micromolar or
a lower concentratioin that inibits neuronal cell death by at least
20 percent.
6. Use of nimesulide for the prepararion of a pharmaceutical
composition for treating Alzheimer's Disease in a subject, wherein
the amount of nimesulide present in the pharmaceutical composition
results in exposure of the affected neurons to a local
concentration of nimesulide of one micromolar or a lower
concentration that inhibits neuronal cell death by at least 20
percent.
7. Use of nimesulide for the preparation of a pharmaceutical
composition for treating epilepsy in a subject, wherein the amount
of nimesulide present in the pharmaceutical composition results in
exposure of the affected neurons to a local concentration of
nimessulide of one micromolar or a lower concentration that
inhibits neuronal cell death by at least 20 percent.
8. Use of nimesulide for the preparation of a pharmaceutical
composition for treating amyotrophic lateral sclerosis in a
subject, wherein the amount of nimesulide present in the
pharmaceutical composition results in exposure of the affected
neurons to a local concentration of nimesuide of one micromolar or
a lower concentration that inhibits neuronal cell death by at least
20 percent.
9. A transgenic mouse carrying a gene encoding the human
cyclcoxygenase-2 gene under the control of a neuron-specific
promoter.
10. The transgenic mouse of claim 9 wherein the level of
cyclooxygenase-2 activity present in some or all neurons of the
mouse is increased.
11. A neuronal cell culture prepared from the transgenic mouse of
claim 9 or 10).
12. Use of nimesulide for the preparation of a pharmaceutical
composition for treating a neurodegenerative condition, wherein the
dosage of nimesulide contained in the pharmaceutical composition
inhibits neuronal cell death by at least 20 percent.
13. Use of nimesulide for the preparation of a pharmaceutical
composition for treating Alzheimer's Disease, wherein thc dosage of
nimesulide contained in the pharmaceutical composition inhibits
neuronal cell death by at least 20 percent.
14. Use of nimesulide for the preparation of a pharmaceutical
composition for treating epilepsy, wherein the dosage of nimesulide
contained in the pharmaceutical composition inhibits neuronal cell
death by at least 20 percent.
15. Use of nimesulide for the preparation of a pharmaceutical
composition for treating amyotrophic lateral sclerosis, wherein the
dosage of nimesulide contained in the pharmaceutical composition
inhibits neuronal cell death by at least 20 percent.
Description
1. INTRODUCTION
[0001] The present invention relates to the use of nimesulide and
structurally related compounds in the prevention and/or treatment
of neurodegenerative conditions such as Alzheimer's Disease. It is
based, at least in part, on the discovery that nimesulide, in
effective concentrations, inhibits cell death.
2. BACKGROUND OF THE INVENTION
2.1. Alzheimer's Disease
[0002] Sporadic Alzheimer's Disease is the major neurodegenerative
disease associated with aging, the risk of developing the disease
rising exponentially between the ages of 65 and 85, doubling every
five years. Histologically, the hallmarks of Alzheimer's Disease
are the deposition of amyloid in senile plaques and in the walls of
cerebral blood vessels; the presence of neurofibrillary tangles,
and neurodegeneration. The etiology of Alzheimer's Disease,
however, is not well understood. Genetic factors have been proposed
to play a role, including trisomy 21, mutations in the amyloid
.beta.-protein precursor ("APP") gene, the presenilin-1 (PS 1) and
presenilin-2 (PS2) genes, and the presence of the apolipoprotein E
type 4 allele (Younkin, 1995, Ann. Neurol. 37:287-288; Lendon et
al., 1997, JAM A 277:825). Several studies have indicated that
.beta.-amyloid induces apoptosis in cultured neurons (Loo et al.,
1993, Proc. Natl. Acad. Sci. U.S.A. 90:7951-7955; Li et al., 1996,
Brain Res. 738:196-204). Such induction may involve the immediate
early gene proteins, c-jun and fos (Anderson et al., 1995, J.
Neurochemistry 65:1487-1498; Anderson et al., 1994, Experimental
Neurology 125:286-295; Anderson et al., 1996, J. Neurosci.
16:1710-1719).
[0003] It has been proposed that apoptosis may be involved in the
pathogenesis of Alzheimer's disease (Smale et al., 1995, Exp.
Neurol. 133:225-230; Anderson et al., 1996, J. Neurosci.
16:1710-1719; Anderson et al., 1994, Exp. Neurol. 125:286-295).
Inflammatory mechanisms have also been implicated; (Pasinetti,
1996, Neurobiol. Ageing 17:707-716) supportive of such a mechanism
are the observations that acute phase proteins are elevated in the
serum of Alzheimer's Disease patients, and are deposited in amyloid
plaques; activated microglial cells tend to localize in the
vicinity of senile plaques, and complement components have been
localized around dystrophic neurites and neurofibrillary tangles
(Aisen and Davis, 1994, Am.J.Psychiatry 151:1105-1113).
Antiinflammatory agents have been suggested as potential
therapeutic agents (Aisen et al., 1996, Dementia 7:201-206; McGeer
et al., 1996, Neurology 47: 425-432). Because they tend to have
fewer adverse side effects, selective inhibitors of the enzyme
cyclooxygenase-2 have been advanced as agents for treating such
inflammation (International Publication No. WO 94/13635 by Merck
Frosst Canada Inc.). Prior to the present invention, however, it
had not been believed that such agents could be used to inhibit
non-inflammatory aspects of neurodegeneration in the context of
Alzheimer's Disease or otherwise.
2.2. Cvclooxvgenase-2
[0004] Cyclooxygenases ("COXs") are enzymes that catalyze the
formation of prostaglandin ("PG")-H.sub.2 from arachidonic acid
(AA). PG-H.sub.2 is further metabolized to physiologically active
PGs (e.g., PG-D.sub.2, PG-E.sub.2 and PG-F.sub.2.alpha.),
prostacyclin (PG-I.sub.2.alpha.) and thromboxanes. Specific PGs
have diverse, often antagonistic effects on different tissues. For
example, PG-I.sub.2 and PG-E.sub.2 are potent vasodilators that may
contribute to the inflammatory response, whereas PG-F.sub.2.alpha.,
is a vasoconstrictor.
[0005] There are two known COX isoforms, COX-1 and COX-2, which,
though physiologically distinct, are similar in amino acid sequence
and enzymatic functions. COX-1 is constitutively expressed at
different levels in different cell types. COX-2, however, is not
constitutively expressed, and is generally undetectable in normal
peripheral tissues (Kujubu et al., 1991, J. Biol. Chem.
266:12866-12872; O'Banion et al. 1992, Proc. Natl. Acad. Sci.
U.S.A. 89:4888-4892). Rather, COX-2 expression is inducible (for
example, by mitogens) and COX-2 mRNA levels have been observed to
rise rapidly in response to inflammatory stimuli such as
interleukin-1.beta. and lipopolysaccharide, and to decrease in
response to glucocorticoids. When subjected to these same factors,
COX-1 mRNA levels remain substantially unchanged, suggesting that
COX-2 is the isoform which mediates inflammation (Cao et al., 1995,
Brain Res. 697:187-196; O'Banion et al. 1992, Proc. Natl. Acad.
Sci. U.S.A. 89:4888-4892).
[0006] Recent evidence suggests that COX-2 may play a role in
mechanisms of cell survival and cell adhesion in peripheral cells
(Lu et al., 1996, Proc. Natl Acad. Sci. U.S.A. 92:7961-7965; Tsujii
et al., 1995, Cell 83:493-501). Tsujii et al. reports that
epithelial cells engineered to express elevated levels of COX-2
were resistant to butyrate-induced apoptosis, exhibited elevated
BCL2 protein expression, and reduced transforming growth factor
.beta.2 receptor levels (Tsuji et al., 1995, Cell 83:493-501). Lu
et al. indicates that non-steroidal antiinflammatory drugs may
induce an apoptotic mechanism involving the COX system.
[0007] The roles of COX-1, COX-2 and PG synthesis in normal brain,
and in the context of Alzheimer's Disease, have not been fully
characterized to date. The importance of PGs in brain physiology
may be independent of inflammatory mechanisms. In the brain, PG
receptors have been identified in the hypothalamus, thalamus, and
limbic system (Watanabe et al., 1989, Brain Res. 478:143-148). PGs
are involved in hypothalamic-pituitary hormone secretion (Kinoshita
et al., 1982, Endocrinol. 110:2207-2209), regulation of temperature
and the sleep-wake cycle (Hayaishi, 1988, J. Biol. Chem.
263:14593-14596). There is recent evidence that COX-2 mRNA is
expressed and regulated in rat brain by synaptic activity and
glucocorticoids (Adams et al., 1996, J. Neurochem. 66:6-13;
Kaufmann et al., Proc. Natl. Acad. Sci. U.S.A. 93:2317-2321;
Yamagata et al., 1993, Neuron 11:371-386). These studies indicate
that COX-2 is regulated as an immediate early gene in the brain,
and suggest that PGs may be important in trans-synaptic signaling
and long-term potentiation. Chang et al. (1996, Neurobiol. of Aging
17:801-808) report that COX-2 mRNA expression is decreased in
Alzheimer's disease.
3. SUMMARY OF THE INVENTION
[0008] The present invention relates to the use of nimesulide and
structurally related compounds in the prevention and/or treatment
of neurodegenerative conditions. It is based, at least in part, on
the discoveries that (i) COX-2 expression in models of
neurodegeneration is increased in neurons rather than glial cells
(consistent with a non-inflammatory mechanism), and (ii) nimesulide
exhibits a neuroprotective effect against .beta.-amyloid induced
neuronal cell death. This latter finding is particularly unexpected
in view of the ability of COX-inhibitors to increase apoptosis of
non-neuronal cells. Without being bound to any particular theory,
it appears that nimesulide inhibits a non-inflammatory mechanism of
neurodegeneration.
4. DESCRIPTION OF THE FIGURES
[0009] FIG. 1. Regional expression of COX-2 mRNA in control
unlesioned male rat brain. COX-2 mRNA expression was assessed by in
situ hybridization and visualized by X-ray autoradiography.
Abbreviations: DG, dentate gyrus; CA1, CA2 and CA3 subregions of
the neuronal pyramidal layer of the hippocampal formation; PAC,
parietal cortex; PYC, pyriform cortex; AC, amygdaloid complex;
THAL, ventroposterior thalamic nucleic. Adapted from plate 24 of
Paxinos and Watson, 1986 (The Rat Brain in Steriotaxic Coordinates,
Academic Press, NY).
[0010] FIG. 2. Maturational regulation of COX-2 mRNA expression in
rat brain. Optical densities were quantified from autoradiographic
images. Abbreviations, as above and as follows: PYC/AC, pyriform
and amygdaloid complex that were quantified for COX-2 mRNA
expression as a single brain region. N=5-8 per time point.
[0011] FIG. 3. Maturational influence on COX-2 mRNA expression
during response to KA-induced seizures. Micrographs were generated
from autoradiographic images. For anatomical distribution of
changes, refer to FIG. 1. Control, unlesioned vehicle-injected
rats; KA 8 H, KA-treated rats 8 hours prior to sacrifice; postnatal
days P-7, P-14, and P-21.
[0012] FIGS. 4A-D. Time course of COX-2 mRNA changes in rat brain
during responses to KA treatment: maturational influences and
regional distribution of changes. Optical densities were quantified
from autoradiographic images. Data are expressed as means.+-.SEM,
N=4=6 per group. *P<0.0l vs 0 H group (saline injected group); 4
h, 8 h, 16 h, 30 h, 120 h, time in hours after onset of KA-induced
seizures.
[0013] FIGS. 5A-D. Maturational influence on the distribution of
COX-2 mRNA expression and induction in rat hippocampus. COX-2 mRNA
in P21 (A,B) and adult (C,D) hippocampal formations as assessed by
in situ hybridization assay and visualized by emulsion
autoradiography using dark-field illumination. In A and C, COX-2
mRNA expression in control rats (vehicle injected); in B and D,
COX-2 mRNA 8 hours after onset of KA-induced seizures. Arrows point
toward the superficial layer of the DG (stratum granulosum). Scale
bar=200 .mu.m.
[0014] FIG. 6. Selective induction of hippocampal COX-2 but not
COX-1 mRNAs during response to KA-induced seizures as assessed by
gel blot hybridization assay. CTL, control saline-injected rats;
KA, KA-treated rats 12 hours post lesioning.
[0015] FIGS. 7A-I. KA-induced COX-2 and apoptosis in adult rat
brain. In (A,B), COX-2 mRNA expression in CA3 hippocampal pyramidal
neurons of control and KA-treated rat, respectively; in (C), CA3
subregion of the hippocampal formation of KA-lesioned rats showing
neurons with apoptotic features (arrows). In (D,E), COX-2 mRNA
expression in pyriform cortex of control and KA-treated rat
respectively. In (F), arrows point toward apoptotic cells of
pyriform cortex of KA-lesioned rats. In (G,H), COX-2 mRNA
expression in cells of the amygdaloid complex of control and
KA-treated rats, respectively. In (I), arrows point toward
apoptotic cells of the amygdaloid complex of KA-lesioned rats.
COX-2 mRNA was assayed by in situ hybridization and visualized by
emulsion autoradiography under dark field illumination. In situ 3'
end-labeling was used to assess apoptotic features following KA
treatment. Scale bar: in A,B,D,E,G,and H=200 .mu.m; in C,F and I=40
.mu.m.
[0016] FIGS. 8A-D. Immunocytochemical evidence of neuronal COX-2
expression/regulation in response to glutamate in vitro. COX-2-like
immunoreactivity in monotypic cultures of rat primary hippocampal
neurons in (A) control and (B) after glutamate exposure (12 hours).
In (C,D) control and glutamate treated cultures immunoreacted with
immunoadsorbed COX-2 antibody, respectively. Scale bar=50
.mu.m.
[0017] FIGS. 9A-C. Effect of nimesulide on endotoxin-mediated
synthesis/secretion of cytokines and nitrites in glia. The effect
of 10.sup.-9 M nimesulide on the synthesis/secretion of (A) TNF,
(B) NO intermediates (Griess reaction) and (C) PGE.sub.2, as
assessed in BV2 mouse immortalized microglial cells. Mean.+-.SEM,
n=8-10 per group. Lipopolysaccharide ("LPS")=5 .mu.g/ml. LPS and
nimesulide were added in combination to cultures; incubation time
was 24 hours. Statistics used ANOVA, p<0.05.
[0018] FIGS. 10A-E. Time course of changes of COX-2 protein in P19
cells during response to conditions leading to apoptotic death. In
(A), quantitative analysis of COX-2 induction in P19 cells, n=4-6
per group, p<0.05 vs. t=0. In (B), changes were assessed by
western analysis, using chemiluminescent detection. In (C,D) the
morphological appearance of apoptotic nuclei in P19 cells was
assessed by Hoechst H33258 24 hours after serum removal (and
replacement with N2 medium). In (E), the electrophoretic profile of
DNA showing DNA laddering degradation was assessed 14 hours after
serum removal (lane 1, control cells at t=0; lane 2, DNA laddering
14 hours after serum removal; lane 3, DNA markers). The COX-2
polyclonal antibody used in these studies was as described in
Section 6. The COX-2 specific antibody recognizes two major bands
having estimated molecular weights of about 70 and 65 kDa in total
homogenates of mouse, rat and human brains.
[0019] FIGS. 11A-E. Regulation of COX-2 during response to
A.beta.1-40 mediated oxidative stress in SH-SY5Y neuronal cells.
(A) Bar graph showing the amount of COX-2 immunoreactivity present
relative to control ("CTL"). The immunoreactivity measurement was
derived from the data shown in (B). (B) Western blot of proteins
prepared from control SH-SY5Y cells or cells exposed to A.beta.
peptides for 48 or 72 hours, reacted with COX-2 antisera. (C)
Results of MTT assays of control or A.beta.-treated SH-SY5Y cells.
(D) The immunoblot shown in (B) stripped and immunoreacted with
actin antisera. (E) SH-SY5Y cultures were examined for apoptotic
mechanisms.
[0020] FIG. 12. Protective effect of nimesulide on A.beta.1-40
mediated neurotoxicity as assessed by MTT assay using SH-SY5Y
neuronal cells. Abbreviations: CTL control; NIM nimesulide; A
B-.beta.-amyloid (1-40). *P<0.01 vs CTL, **P<0.05 vs.
CTL.
[0021] FIG. 13. Micrographs showing induction of COX-2
immunoreactivity in temporal cortex of AD and age-matched
neurological controls. The micrographs show a selective induction
of COX-2 immunostaining in neuron-rich grey matter ("gm") but not
glia-rich white matter ("wm") regions.
[0022] FIGS. 14A-D. COX-2 immunostaining of neurons in frontal
cortex of AD brain and co-localization of COX-2 immunostaining with
AD plaques in frontal cortex of AD brains. In (A,B), immunostaining
of COX-2 neurons of AD are shown (A, low power; B, high power
magnification). In (C), immunostaining of diffuse plaques. In (D),
COX-2 in neuritic plaques as assessed by A.beta. immunostaining on
adjacent tissue sections.
[0023] FIGS. 15A-D. COX-2 expression in AD brain frontal cortex.
(A) Northern blots of COX-2 and COX-1 mRNA in AD frontal cortex
relative to controls quantitative analysis of data is in (B). (C)
Western blots of COX-2 protein in AD frontal cortex and control;
quantitative analysis of data is in (D).
[0024] FIG. 16. COX-2 mRNA regulation in neurons of the anterior
and posterior horn of the spinal cord of ALS and neurological
controls. Autoradiographic images were visualized by X-ray film.
The arrow pointed toward the ventral horn shows intense induction
of COX-2 mRNA signal in the ALS case.
[0025] FIGS. 17A-D. COX-2 mRNA elevation in a biopsy of human
cortical epileptic foci as assessed by in situ hybridization assay.
Autoradiographic images were visualized by X-ray film. Arrow
pointed toward the cortical layers showing intense COX-2 mRNA
signal in epileptic brain (A) versus control (B). Parts (C) and (D)
show analysis of compiled data relating to COX-2 and COX-1 mRNA
levels.
[0026] FIG. 18. Potentiation of cyclooxygenase and peroxidase
activities by aggregated A.beta. peptides.
[0027] FIGS. 19A-B. Transient expression of COX-2 in neuronal cells
potentiates A.beta.-mediated impairment of redox activity. SH-SY5Y
neuronal cells were transfected with either the human COX-2 (A) or
COX-1 (B) gene and treated with A.beta..sub.25-35, and then redox
activity was assessed by MTT assay.
[0028] FIGS. 20A-B. Generation of Transgenic Mice Expressing COX-2.
(A) In situ hybridization demonstrates COX-2 mRNA expression in
TgNHC32 mice but not wild-type (mice one month old). (B) Regional
expression of hCOX-2 mRNA in NHC32 and NHC5 relative to wild-type,
as quantified using Bioquant image-analysis.
[0029] FIG. 21. hCOX-2 over-expression potentiates A.beta.-mediated
oxidative impairment in primary mouse neuronal cultures.
[0030] FIG. 22. Nimesulide protects B12 neuronal cells against
glutamate mediated oxidative stress.
[0031] FIG. 23. Control studies for FIG. 22. Dose (A) and time
course (B) association with glutamate mediated oxidative impairment
as measured by LDH assay. (C) demonstrates lack of toxicity of
nimesulide to B12 cultures.
5. DETAILED DESCRIPTION OF THE INVENTION
[0032] The present invention relates to the use of the
cyclooxygenase-2 ("COX-2") selective inhibitor, nimesulide, and
structurally related compounds in the prevention and/or treatment
of neurodegenerative conditions. It is based, at least in part, on
the discoveries that COX-2 expression increased in parallel with
neuronal lesions produced by kainic acid and with induction of
neuronal apoptosis in an established in vitro system (see Sections
6 and 8, below). Contrary to reports in the prior art that COX-2
plays a primary role in inflammation and may be involved in an
inflammatory component of neurodegeneration (for example, in the
context of Alzheimer's Disease), it has further been discovered
that in the human brain, COX-2 expression appears to be restricted
to neurons rather than to glial cells (whereas expression in glial
cells would be expected if an inflammatory mechanism were
operative: see Section 9, below). A correlation between COX-2
expression and the characteristic pathological change associated
with Alzheimer's Disease, amyloid plaque, has been observed (see
Section 9). Further, it has been demonstrated that nimesulide
exerts a neuroprotective effect against .beta.-amyloid induced cell
death in neuronal cultures. In view of these findings, according to
the invention, nimesulide and related compounds may be used to
intervene in the process of apoptotic neuronal cell death
associated with Alzheimer's Disease and other neurodegenerative
conditions. Without being bound to any particular theory, the
action of nimesulide and related compounds may be mediated by the
reduction of the apoptotic effects of oxidative stress.
[0033] The term "nimesulide", as used herein, refers to a compound,
4-nitro-2-phenoxymethanesulfonaninide, having a structure as set
forth for Formula I below. 1
[0034] Compounds which are considered structurally related are
compounds which have a similar bicyclic structure and which
selectively inhibit COX-2. For example, substituents, analogs, and
enantiomers of nimesulide are considered to be structurally related
compounds. As a further example, a structurally related compound
may compete with nimesulide for binding to COX-2, or may bind to
substantially the same location of COX-2, as determined
crystallographically. Moreover, nimesulide, conjugated to another
compound, is also considered to be a structurally related compound
as defined herein.
[0035] Nimesulide, or a structurally related compound, may be
administered so as to provide an effective concentration in the
nervous system of the subject being treated. An effective
concentration is defined herein as that concentration which
inhibits neuronal cell death by at least 20 percent. In specific,
nonlimiting embodiments of the invention, the concentration of
nimesulide is at least 1 nanomolar, and preferably at least 1
micromolar in the location of neuronal cells which are desired to
be treated. Desirably, the concentration of nimesulide is less than
10.sup.-3-molar to avoid toxicity. In one such specific embodiment,
where the neurodegenerative condition to be treated is Alzheimer's
Disease, the concentration of nimesulide in the hippocampal
formation of the subject is at least 1 picomolar, preferably at
least 1 nanomolar, and more preferably at least 1 micromolar. The
corresponding serum concentrations may be at least 10 picomolar,
preferably at least 10 nanomolar, and more preferably at least 10
micromolar. Equivalent amounts of structurally related compounds
(adjusted to compensate for differences in potency) may also be
used.
[0036] Nimesulide or a structurally related compound may be
administered in any manner which achieves the desired effective
concentration. For example, suitable routes of administration
include oral, intravenous, subcutaneous, intramuscular, transdermal
and intrathecal routes.
[0037] Nimesulide or a structurally related compound may be
comprised in a suitable pharmaceutic carrier. Formulations may
provide for sustained release.
[0038] For example, but not by way of limitation, nimesulide may be
administered orally at doses of 2-800 mg/day, preferably 50-400
mg/day, and most preferably 200 mg/day.
[0039] Neurodegenerative conditions which may be treated according
to the invention include, but are not limited to, Alzheimer's
Disease, Parkinson's Disease, seizure-associated neurodegeneration,
amyotrophic lateral sclerosis, spinal cord injury and other
diseases wherein the condition is not conventionally regarded as an
inflammation-mediated or autoimmune disorder.
6. EXAMPLE
Maturational Regulation and Regional Induction of Cyclooxygenase-2
in rat Brain
[0040] 6.1. Materials and Methods
[0041] Animals and excitotoxic lesions. Male adult Sprague/Dawley
rats of different ages were maintained in a controlled light and
temperature environment, with food and water ad libidum. In adult
rats (250-300 g), hippocampal excitotoxic lesions were induced by
subcutaneous injection of kainic acid ("KA"; 10 mg/kg, Sigma).
Because KA uptake is higher in young rats relative to adults
(Berger et al., 1986, in Schwartz and Ben-Ari, Advances in
Experimental Medicine and Biology, Plenum, N.Y., pp. 199-209), KA
doses were adjusted to produce maximal excitotoxicity without
reaching lethal doses (from 2 mg/kg at postnatal day P-7 to 6 mg/kg
at postnatal day P25). Saline injected rats were used as controls
(0 hour time point).
[0042] COX-1 and COX-2 cDNA probes. Bluescript plasmid (Stratagene)
containing the full length rat COX-1 cDNA (2.7 kb) was linearized
by digestion with ClaI; PCRII plasmid (Invitrogen) containing the
coding sequence for rat COX-2 (1.8 kb) was linearized by digestion
with PflMI (Feng et al.,1993, Arch. Biochem.
[0043] Biophys. 307:361-368). Linearized plasmids were purified
using Elu-Quick (Schleicher & Schuell) after agarose gel
electrophoresis.
[0044] Int situ hybridization. At various intervals after the onset
of KA-induced seizures, the rats were sacrificed, and the brains
quickly removed, rinsed in cold phosphate buffer (PBS, 10 mM, pH
7.4) and immersed in methylbutane at -25.degree. C. for three
minutes. The brains were sliced into 10 micrometer sections,
frozen, and the resulting frozen sections were mounted on
polylysine-coated slides and stored at -70.degree. C. For
immunocytochemistry ("ICC") or in situ hybridization ("ISH"),
frozen sections were post-fixed in PBS containing 4 percent
paraformaldehyde (30 minutes at room temperature) and then rinsed
in PBS. For ISH, tissue sections were rinsed in 0.1M
triethanolamine ("TEA"), pH 8.0, incubated in acetic anhydride
("AAH"; 0.25% v/v in TEA, 10 minutes) and rinsed in TEA and PBS.
Following AAH treatment, tissue sections were hybridized with
[.sup.35S]-cRNA probes (0.3 .mu.g/ml, 2.times.10.sup.9 dpm
.mu.g-.sup.-1) made from COX-2 linearized cDNA transcription
vectors (Feng et al., 1993, Arch. Biochem. Biophys. 307:361-368).
Sense strand hybridization was used as a control and gave negative
results. Following hybridization for 3 hours at 50.degree. C.,
stringent washes (0.1.times.SSC at 60.degree. C.) and dehydration,
slides were exposed to X-ray film for seven days for
quantification. Slides were then exposed to NTB-2 emulsion (Kodak,
Rochester, N.Y.) for microscopic analysis of COX-2 mRNA
distribution. Following development, tissue sections were
counterstained with cresyl violet. Film autoradiograms were
analyzed quantitatively using an image analysis system with
software from Drexel University (Tocco et al., 1992, Eur. J.
Neurosci. 4:1093-1103). Statistics were calculated by ANOVA
followed by additional posthoc analysis.
[0045] In situ end labeling. In parallel studies,
paraformaldehyde-fixed brain tissue sections were dehydrated, air
dried and incubated with dATP, dCTP, dGTP (0.2 mM), dTTP (13
.mu.M), digoxigenin-11-dUTP and DNA polymerase I (Boehringer
Mannheim) at 10 units/100 .mu.l at 37.degree. C. for 2 hours. The
reaction was stopped by addition of 20 mM EDTA, pH 8.0. Sections
were incubated at room temperature overnight with an
alkaline-phosphatase-conjugated digoxigenin antibody (Genius
System, Boehringer Mannheim) diluted 1:200 in 5 percent sheep serum
diluted in 150 mM NaCl, 100 mM TRIS-HCl, pH 7.5. Colorimetric
detection with nitro blue tetrazolium and
5-bromo-4-chloro-3-indolyl-phosphate was performed with the Genius
System by the manufacturer's protocol (Sakhi et al.,Proc. Natl.
Acad. Sci. U.S.A. 91:7525-7529).
[0046] Northern blot hybridization assay. RNA extraction was
performed as follows. After sacrifice, rat brains were dissected
and stored at -75.degree. C. prior to processing. Total RNA was
extracted from pools of hippocampal tissue (Pasinetti et al.,1994,
J. Comp. Neurol. 339:387-400). Briefly, tissues were homogenized
for 1 minute in 4 M guanidinium thiocyanate, 25 mM sodium citrate
(pH 7.5), 0.5% sarcosyl and 0.1 M .beta.-mercaptoethanol, in a
final volume of 0.5 ml. After acidified phenol/chloroform
extraction and ethanol precipitation, the RNA pellet was rinsed
consecutively with 70% and 100% ethanol. The purified RNA was then
dissolved in 0.5% sodium dodecyl sulfate ("SDS") and stored at
-75.degree. C. Total RNA was quantified in a UV spectrophotometer.
Total RNA (5 .mu.g) from tissue was electrophoresed on denaturing
(0.2M formaldehyde) agarose gels and transferred to a nylon
membrane (Nylon 66 plus; Hoeffer, San Francisco Calif.) in
2.times.SSC. Blot hybridization was carried out with 10.sup.6
cpm/ml of antisense COX-1 or COX-2 [.sup.32P]-cRNA probes in 50%
formamide, 1.5.times.SSPE, 1% SDS, 0.5% dry milk, 100 .mu.g/ml
yeast total RNA and 500 mg/ml salmon sperm DNA at 53.degree. C. for
about 15 hours. Blots were washed to a final stringency of
0.2.times.SSC, 0.2% SDS at 72.degree. C. Blots were exposed to
Kodak X-ray film (XAR-5) with intensifying screens at -70.degree.
C.
[0047] Cell cultures. Hippocampal neuron cultures were derived from
embryonic rat brain. E16-E18 embryos were dissected in Hank's
balanced salt solution and cultured (Pasinetti et al.,1994, J.
Comp. Neurol. 339:387-400; Peterson et al., 1989, Dev. Brain Res.
48:187-195). Culture media were changed every 3 days. For glutamate
neurotoxicity studies, eight day old cultures were treated with
glutamate (250 .mu.M, Sigma) in the presence of 2.4 mM calcium ion
and 0.8 mM magnesium ion. After 6 hours of glutamate exposure,
culture medium was replaced with fresh medium; COX-2-like
immunoreactivity was assessed in neurons 12 hours later.
[0048] Immunocytochemical detection of COX-2 in monotypic cultures
of primary rat neurons. Control and glutamate treated cultures were
post-fixed in PBS containing 4% paraformaldehyde (30 minutes, room
temperature), rinsed in PBS, pre-treated with normal serum and
incubated overnight at 4.degree. C. with primary antibodies. COX-2
antisera (rabbit IgG) was raised against a synthetic peptide
(CNASASHSRLDDINPT; SEQ ID NO:1) encompassing the C-terminal region
of the murine COX-2. The antisera reacts with human and rat COX-2
but not with COX-1, as assessed by Western blot analysis.
Vectastain ABC kit (Vector, Burlingame, Calif.) was used in
subsequent steps to complete the diaminobenzene staining (Pasinetti
et al.,1994, J. Comp. Neurol. 339:387-400). Immunoadsorption of
COX-2 antisera with synthetic COX-2 peptides controlled for
specificity; adsorption was carried out overnight at 4.degree. C.
with synthetic COX-2 peptides at 30 .mu.g/ml.
[0049] 6.2. Results
[0050] COX-2 expression in adult rat brain. FIG. 1 shows by ISH
that the regional distribution of COX-2 mRNA was most notable in
limbic structures, but was also present in neocortex (consistent
with the reports of Kaufmann et al., Proc. Natl. Acad. Sci. U.S.A.
93:2317-2321 and Yamagata et al., 1993, Neuron 11:371-386). In the
hippocampal formation, COX-2 mRNA was selectively expressed in
cells of the granule and pyramidal neuron layers. COX-2 mRNA
expression was also found in the outer layer of the parietal
cortex, the pyriform cortex and cells of the amygdaloid
complex.
[0051] Maturational regulation of hippocampal COX-2 mRNA
expression. ISH results indicated that during maturation, COX-2
MnRNA expression is differentially regulated in subsets of cells of
neuronal layers of the hippocampal formation (FIG. 2, FIG. 3 top).
From postnatal days P7-P14, COX-2 mRNA showed greater than two-fold
increase in the granule cell layer of the dentate gyrus and in the
CA3 subdivision of the pyramidal cell layer (p<0.001, FIG. 2).
Though the expression of COX-2 mRNA was lower in the other brain
regions examined, the pattern of maturational expression was
similar (FIG. 2). By postnatal day P21, COX-2 mRNA expression
approximated adult levels in all subregions examined (FIG. 2, FIG.
3 top).
[0052] Response to KA-induced seizures. To further explore
maturational regulation of COX-2 in brain, COX-2 mRNA expression
during responses to KA-induced seizures was examined postnatally.
Despite intense seizure activity after KA treatment, no detectable
change of COX-2 mRNA expression was found in any brain region
examined at P7 (FIG. 3, FIG. 4A). Changes in COX-2 mRNA expression
at 120 hours post-KA treatment in the P7 group indicate
developmental maturation rather than response to KA toxicity (FIG.
4A). In contrast to the P7 group, at P14 and P21 COX-2 mRNA
increased within 4-8 hours after onset of KA-induced seizures in
all the hippocampal subregions examined (FIGS. 4B and 4C). The
level of COX-2 mRNA expression returned toward control levels
within 120 hours after treatment in P14, P21 and adult rat
brain.
[0053] Within the dentate gyrus, control COX-2 expression in P14
and P21 rats was asymmetric, selectively localized to the more
superficial neurons of the stratum granulosum rather than the
deeper granule cells of the dentate gyrus blade (FIG. 5A). In
response to KA treatment, COX-2 mRNA induction showed similar
asymmetry of expression (FIG. 5B). In contrast, the asymmetry
within the dentate gyrus was less notable in control animals and
after KA-induction of the adult group (FIGS. 5C and 5D).
[0054] In parallel studies, northern blot hybridization of total
RNA from hippocampus of adult rats 12 hours after KA-induced
seizures confirmed COX-2 mRNA induction (FIG. 6). No detectable
induction of COX-1 mRNA was found in the same rat brain (FIG.
6).
[0055] KA-induced COX-2 and apoptosis in adult rat. By 8 hours
after onset of KA-induced seizures, COX-2 mRNA induction in cells
of the CA3 region of the hippocampal formation (FIG. 7B), pyriform
cortex (FIG. 7E) and amygdaloid complex (FIG. 7H) of the adult
brain paralleled temporally and overlapped anatomically the onset
of apoptosis as assessed by in situ end-labeling in the same brain
regions (FIG. 7C, CA3 regions of the hippocampus; FIG. 7F, pyriform
cortex; FIG. 7I, amygdaloid complex). Cellular COX-2 mRNA
expression in adult, control (FIGS. 7A, 7D and 7G) and KA-treated
(FIGS. 7B, 7E and 7H) rats was identified by emulsion
autoradiography using ISH assays.
[0056] Immunocytochemical evidence of neuronal COX-2
expression/regulation in response to glutamate in vitro. Primary
cultures of rat hippocampal neurons were exposed to glutamate in
vitro. At baseline, constitutive COX-2 expression was demonstrated
by immunocytochemistry (FIG. 8B). Twelve hours after exposure to
glutamate, an increase in COX-2 immunoreactivity was observed which
coincided with marked reduction in the number of neurons (FIG.
8D).
[0057] COX-2 expression in human epilepsy. Increased expression of
COX-2 mRNA was detected by ISH in a biopsy of human brain at
epileptic foci (FIGS. 17A-D). It was found that COX-2 mRNA, but not
COX-1 mRNA, was significantly elevated in the cortex of epileptic
patients. the p vlaue was <0.05.
7. EXAMPLE
Nimesulide Suppresses Cytokine and Nitrite Production in Microglia
Cultures
[0058] In experiments conducted in vitro, nimesulide at low
concentration (1 nanomolar) was found to be effective in the
suppression of endotoxin-mediated induction of tumor necrosis
factor ("TNF") production by immortalized brain-derived microglia
(BV-2) and astrocytes (FIG. 9A). In parallel, nimesulide was
equally effective in blocking nitrite production (Griess reaction;
FIG. 9B). This latter observation is particularly relevant in view
of evidence showing that blockade of neuronal nitric oxide
("NO")-synthase protects against glutamate neurotoxicity.
Nimesulide was also found to be effective in blocking
endotoxin-mediated induction of prostaglandin PGE.sub.2) in
brain-derived microglia (FIG. 9C).
8. EXAMPLE
COX-2 Expression in Apoptotic Cells
[0059] The regulation of COX-2 expression was studied using an
established in vitro model of apoptosis. Specifically, the
regulation of COX-2 was studied in P19 embryonic carcinoma cells
during responses to serum deprivation. Under such conditions, the
P19 cells underwent apoptotic cell death, showing characteristic
DNA fragmentation and nuclear morphology. As shown in FIG. 10,
using this system, coincidental onset of apoptosis and elevation of
COX-2 expression was observed.
[0060] These studies were extended to the human neuronal cell line
SH-SY5Y. .beta.-amyloid has been demonstrated to play a role in
inducing neurodegeneration in SH-SY5Y cells (Oda et al., 1995,
Alzheimers Res. 1:29-34). SH-SY5Y cells were treated with synthetic
aggregated A.beta..sub.1-40 peptides at a concentration of 20 .mu.M
for various periods of time. Western blot analysis was performed of
cell extracts, using COX-2 antisera or actin antisera as control
(FIGS. 11B and D, respectively). In FIG. 11A, COX-2 protein from
control or A.beta.-treated SH-Sy5Y neuronal cells was quantified
from the immunoblots shown in FIG. 11B (at the 72 hr. time point;
only the 70 kDa mw species being quantified). COX-2 signal was
abolished by immunoadsorption of COX-2 antisera with purified human
recombinant COX-2 ("hrCOX-2") peptides. FIG. 11C shows that
diminished cell redox activity was observed 72 hours after
treatment with aggregated A.beta..sub.1-40 (at 20 .mu.m), as
assessed by MTT assay in parallel cultures. Bar graphs represent
the mean.+-.SEM, n=4-5 per group, p<0.05 (t-test). COX-2
integrated optical densities were analyzed from digitized images
using a Bioquant image analysis (Biometrics, Nashville, Tenn.). It
was also found that COX-2 expression occurred in parallel to DNA
laddering (FIG. 11E), an index of apoptosis. FIG. 12 shows that
nimesulide at 10.sup.-6 and 10.sup.-9 molar was able to block
A.beta.1-40 toxicity, as assessed by the MTT assay.
[0061] Interestingly, when aggregated synthetic A.beta..sub.1-40
peptides (150 .mu.M) were coincubated with purified hr-holoCOX-2
(COX-2 plus heme-cofactor, 50 nM) for 16 hours, at 37.degree. C. in
1X PBS, and cyclooxygenase and peroxidase activities were measured
(Murphy et al., 1989, Neuron 2:1547-1558) cyclooxygenase and
peroxidase activities increased (relative to activity levels of
enzyme in the absence of A.beta.; FIG. 18). Cyclooxygenase and
peroxidase levels in the absence of heme gave negative results.
9. EXAMPLE
COX-2 Expression in Alzheimer's Disease
[0062] The expression of COX-2 in normal human brains and in brains
of Alzheimer's Disease ("AD") patients was studied.
Immunocytochemical data indicates that COX-2 expression is
primarily localized to cells with neuronal morphology in human
brain; minimal localization of COX-2 immunostaining in cells with
glial morphology was found in any region examined. These results
tend to suggest a role for COX-2 in a non-inflammatory function.
While the immunocytochemical signal for COX-2 was at the limit of
detection in temporal cortex of neurological control cases, COX-2
showed elevation in AD brain (FIG. 13A, 13B) The selective
induction of COX-2 immunoreactivity in grey matter is consistent
with the findings showing neuronal induction of COX-2 in rat brain
during responses to lesions leading to neuronal death (see Section
6).
[0063] The cellular immunodistribution of COX-2 in AD brain was
also explored. It was observed that neuronal COX-2 is not only
localized to the perikarya (FIG. 14A, but is found in neuronal
projections as well (FIG. 14B). Moreover, intense immunostaining
was also found in diffuse plaques (FIG. 14C) and in AD neuritic
plaques identified by A.beta. immunostaining on adjacent tissue
sections of the hippocampal formation. These findings suggest a
role for COX-2 in mechanisms of neuronal death or survival.
[0064] Studies were performed to quantify COX-2 expression in
brains of AD and age-matched controls. We used quantitative
dot-blot analysis and chemiluminescence detection. Western analysis
was used for qualitative assessment. A greater than 2-fold
elevation of COX-2 content was found in hippocampal homogenates of
AD brains compared to neurological age-matched controls.
[0065] Because of the immunocytochemical evidence showing COX-2
immunoreactivity in AD plaques, levels of COX-2 and total A.beta.
in AD cases were compared. A direct correlation was found.
[0066] COX-2 mRNA and protein were found to be increased in AD
brain frontal cortex. FIG. 15A shows Northern blots of total RNA
from neurological control (C) and AD frontal cortex hybridized to
[32p] COX-2 and COX-1 mRNAs. The mRNA species detected by COX-2 and
COX-1 cRNA probes represent an RNA of the same size found in
peripheral tissues. FIG. 15B shows quantified results of the
Northern studies. Specifically, COX-2 mRNA prevalence was increased
in the frontal cortex of AD vs control, where the presence of equal
amounts of RNA in both lanes was confirmed by normalization to 28S
rRNA in the hybridization membrane, and complete transfer of RNA to
the membrane was evidenced by the absence of 28S and 18S rRNAs in
the gel post transfer. Integrated optical densities were analyzed
from digitized images using a Biquant image analysis. Bar graphs
represent n=9 per group, and the results were significant by a
t-test value of p<0.05.
[0067] Similarly, when Western blot analysis was performed of AD
frontal cortex vs. controls, the amount of COX-2 protein was found
to increase. FIG. 15C shows the results of such a western blot.
Lanes 1 and 2 show the COX-2 protein content of the same tissues
uses for Northern analysis. Lanes 3-4 show the absence of COX-2
bound when COX-2 antisera was immunoadsorbed using hrCOX-2
peptides, demonstrating the specificity of the signal.
[0068] The expected 70 kDa new species of hrCOX-2 (lane 5) also was
absent when antisera was preadsorbed with hr COX-2 peptides. These
results are quantified in FIG. 15D (with respect to the 70 kDa
species). Further, the immunoblots were stripped and imnnunoreacted
with actin antisera to verify the specificity of changes (actin
values: control group 100.+-.8; AD 94.+-.7% of control Bar graphs
represent n=9 per group; t-test value is p<0.05). The inset in
FIG. 15D shows a correlation of COX-2 content and the number of
plaques per mm.sup.2, n=8, r=0.72, p<0.03. COX-2 and actin
integrated optical densities were analyzed from digititzed images
using Biquant image analysis.
10. EXAMPLE
COX-2 Expression in Amyotrophic Lateral Sclerosis
[0069] In situ hybridization has demonstrated increased COX-2 mRNA
in the anterior horn cells of spinal cords of patients suffering
from amyotrophic lateral sclerosis ("SACS"); FIG. 16.
11. EXAMPLE
Transient Expression of COX-2 In Neuronal Cells Potentiates A.beta.
Mediated Impairment of Redox Activity
[0070] The role of COX-2 in neuronal death and/or survival was
studied in cultured human SH-SY5Y neuronal cells. Cells were
transiently transfected with a mammalian expression vector
(pRc/CMV/hCOX, Invitrogen) containing either a filll length human
(h) COX-2 cDNA (pRc/CMV/hCOX-2), or a bacterial chloramphenicol
acetyltransferase (CAT) gene (pRc/CMV2/CAT). Following A.beta.25-35
treatment, impairment of redox activity as assessed by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetraxolium bromide
(MTT)-assay (FIG. 19). Overexpression of hCOX2 in SH-SY5Y cells
transfected with pRc/CMV/hCOX-2 was confirmed immunocytochemically
in a parallel tissue culture chamber slide using an .alpha.-COX-2
antibody as previously described. In control cells transfected with
pRc/CMV2/CAT, the same .alpha.-COX antibody generated minimal
immunocytochemical signal. We found that transient overexpression
of hCOX-2 in SH-SY5Y neuronal cells potentiated A.beta.25-35 (25
.mu.M, 48 hrs treatment) mediated impairment of redox activity when
compared to SH-SY5Y cultures transfected with control vector
(.+-..+-.p<0.01 vs A.beta./control vector; *p<0.001 vs CTL.
These data are consistent with evidence showing induction of COX-2
mRNA coincidentally with impairment of redox activity in SH-SY5Y
neuronal cells (FIG. 11).
12. EXAMPLE
Experiments Using COX-2 Transgenic Mice
[0071] Based on the evidence that overexpression of COX-2 in
neuronal cells may potentiate A.beta. -mediated redox impairment
and the observation that neuronal COX-2 is elevated in AD brain and
in experimental neurodegeneration, we generated a transgenic mouse
model with neuronal overexpression of human (h) COX-2. To obtain
overexpression of hCOX-2 in neurons, a hybrid gene was prepared in
which the expression of a cDNA sequence containing the entire
hCOX-2 coding region is regulated by the rat neuron-specific
enolase (NSE) promoter.
[0072] We obtained four lines with high expression of hCOX-2 mRNA
expression. In one of them (NHC32), COX-2 mRNA was assessed by in
situ hybridization analysis (FIG. 20A). We found that in NHC32
mouse line, has high levels of hCOX-2 mRNA in the hippocampal
formation, cerebral cortex and in other neuronal layers (FIG. 20B).
No white matter level of HCOX-2 mRNA were found using combined
immunocytochemistry for NSE and hCOX-2 in situ hybridization on the
same tissue section, we confirmed the selectivity of hCOX-2
transgene expression to neuronal cells.
[0073] It has further been shown, using neuronal cultures derived
from the transgenic mice, that hCOX-2 overexpression potentiated
A.beta. mediated response. As depicted in FIG. 21, studies showed
that primary neuronal cultures derived from transgenic mice with
neuronal COX-2 overexpression (NHC32, n=3), are most susceptible to
aggregated A.beta.25-35 peptides mediated impairment of redox
activity (MTT assay, A.beta.25-35 25 .mu.M for 48 hrs), when
compared to equally treated neuronal cultures derived from wild
type/control littermates (n=4). Moreover, when examined
morphologically, A.beta.25-35 treated neurons derived from
transgenic hCOX-2 mice revealed an intensified regression of
neuronal processes (B, inset) when compared to wild type control
A.beta.25-35 treated neuronal cultures (A, inset).
13. EXAMPLE
Nimesulide Protects B12 Neuronal Cells Against Glutamate
Toxicity
[0074] We examined the role of nimesulide, indomethacin and NS398
(another COX-2 preferential inhibitor) on glutamate mediated
toxicity. In this study we used B12 neuronal cells; toxicity was
assessed by amount increase of lactate dehydrogenase LDH levels in
the conditioned medium after treatment. LDH is an index of cell
toxicity and its elevation is generally accepted as marker of
neurotoxicity. As shown in FIGS. 22-23, we found that nimesulide
protected against glutamate toxicity (10 mM) at 10-6, 10-9 and
10-12 M with pretreatment (24 hrs before) and cotreatment (with
glutamate for 24 hrs). Indomethacin, protected at 10-6 M and lost
its protective effect at 10-9 and 10-12 M. Marginal protection of
glutamate mediated toxicity by indomethacin was observed at 10-9 M
(pretreatment 24 hrs). No protection was observed with NS398
pretreatment. In the pretreatment condition drugs remained in the
media during glutamate treatment.
[0075] Various publications are cited herein, the contents of which
are hereby incorporated in their entireties.
Sequence CWU 1
1
1 1 16 PRT Artificial Sequence synthetic peptide 1 Cys Asn Ala Ser
Ala Ser His Ser Arg Leu Asp Asp Ile Asn Pro Thr 1 5 10 15
* * * * *